Drug discovery is a costly affair where one of the major expenses in terms of money and time is the in vivo studies. In order to reduce these costs a large number of in vitro models are developed and applied as filters to select the most suitable compounds for the in vivo studies. However, in vitro models are often too simplified and may as such be misleading in the decision-making process. Hence, there is a demand for intermediate models that are more reliable than in vitro models and at the same time faster and cheaper than traditional vertebrate in vivo models.
The absorption of drugs via the oral route is a subject of intense and continuous investigation in the pharmaceutical industry since good bioavailability implies that the drug is able to reach the systemic circulation. Oral absorption is affected by both drug properties and the physiology of the gastrointestinal tract, including drug dissolution from the dosage form, the manner in which drug interacts with the aqueous environment and membrane, permeation across membrane, and irreversible removal by first-pass organs such as the intestine, liver, and lung (Martinez and Amidon, 2002).
The intestine, in addition to the liver, is an important tissue that regulates the extent of absorption of orally administered drugs, since the intestine and liver are involved in first-pass removal (Gibaldi et al., 1971; Rowland, 1972). The majority of drug absorption occurs at the small intestine where the presence of villi and microvilli markedly increases the absorptive area. The duodenum and jejunum possess the greatest surface areas due to the highest concentration of villi and microvilli in these regions, and surface area is least for the ileum (Magee and Dailey, 1986).
The intestine is unique in that the intestinal venous blood constitutes the majority of the blood supply to the liver, accounting for 75% of total liver blood flow. Drugs that are absorbed by the intestine, will reach the liver and the lung, for metabolism and for elimination (Gugler et al., 1975; Xu et al., 1989; Hirayama et al., 1990).
Since a large number of factors will affect the function of the intestine and thus also the uptake of orally administered drugs (Xu et al., 1989; Hirayama and Pang, 1990, Chen and Pang, 1997, Welling, 1984; Kimura and Higaki, 2002) there is a high demand on an intestinal model to be representative of the vertebrate intestinal function but also highly reproducible.
Because of the significance of the intestine as an important first pass organ after oral drug intake, high-throughput in vitro systems have been developed to assess intestinal absorption, metabolism, and excretion for the prediction of the bioavailability of a given chemical substance. Gene expression systems (Smit et al., 1998b; Cvetkovic et al., 1999; Gotoh et al., 2000; Shitara et al., 2002) provide direct information on the involvement of individual transporters or enzymes. Then there are the intestinal membrane segments/preparations (Wilson and Treanor, 1975; Hopfer et al., 1976; Lasker and Rickert, 1978; Johnson et al., 2001), cells (Koster and Noordhoek, 1983; Traber et al., 1991), everted sacs (Munck, 1965; Barr and Riegelman, 1970), and the Ussing chamber (Fiddian-Green and Silen, 1975). For flux measurements, a donor compartment is used for drug administration and a receiving compartment is used for sampling. With drug given to the mucosal side, sampling allows the examination of drug absorption, metabolism, and efflux as well as entry into the basolateral compartment. Moreover, a drug may be given at the serosal compartment to ascertain the net flux from the basolateral side to the mucosal lumen.
A popular in vitro system is the Caco-2 cell line, derived from human colon carcinoma cells (Hidalgo et al., 1989). A drawback is the existence of a unstirred water layer that may pose as a barrier for lipophilic drug transport (Hidalgo et al., 1991). The development of the Caco-2 penetration model has greatly facilitated progress and led to the testing of diverse drug classes as Pgp substrates (Burton et al., 1993).
The in situ vascularly perfused rat small intestine preparation is a useful preparation for studying the disposition of both orally and systemically administered agents (Windmueller and Spaeth, 1977, and Doherty and Pang, 2000). In this preparation, the native architecture of the small intestine is maintained with respect to the circulation such that the extents of metabolism, absorption, and secretion can be studied simultaneously. The technique allows for single-pass or recirculating experiments involving systemic or luminal drug administration, including luminal administration in closed loops or segments.
In vivo techniques exist for the study of intestinal drug absorption. The Doluisio method entails use of an in situ rat gut technique for drug administration into the lumen (Doluisio et al., 1969). In some rat preparations, the inflow and outflow of a select segment were monitored for drug disappearance, and arterial blood was sampled and the volume of blood was replenished by transfusion (Barr and Riegelman, 1970). Some studies involve luminal instillation of drug to selected or closed segments of rats (duodenum, jejunum, or ileum) (Hirayama et al., 1990) or humans (Gramatté and Richter, 1994).
The alimentary tract of insects is divided into three main regions: the foregut, midgut and the hindgut. Both the foregut and the hindgut are of ectodermal origin while the midgut is of endodermal origin. The cells in the foregut are usually flattened and are not involved in absorption or secretion. Part of the foregut is a crop which is a storage organ and in most insects an extensible part of the foregut. The effectiveness of the crop as a storage organ (especially for fluid feeding insects) is underlined by its impermeability to hydrophilic molecules. In orthopteroid insects the crop is developed into a grinding apparatus with strong cuticular plates or teeth which brake up the food.
The cells of the midgut are actively involved in enzyme production as well as in absorption of nutrients. The majority of cells are so called principal cells. These cells are tall columnar cells and similar to the enterocytes in the vertebrate intestine, these cells exhibit huge numbers of microvilli towards the luminal side. This morphology strongly indicates that the principle cells are very metabolically active and are main players in nutrient absorption and secretion of digestive enzymes (Lehane and Billingsley, 1996). Also similar to vertebrate intestine there are proliferative zones or nidi in the midgut, with the same function as stem cells in vertebrate intestine (Illa-Bochaca and Montuenga, 2006). Since the principal cells in the insect midgut, similar to vertebrate enterocytes, have a limited life span there is a need for a high regenerative capacity. The undifferentiated cells, daughter cells from the nidi, give rise to both differentiated columnar principal cells and endocrine cells.
The hindgut is usually differentiated into pylorus, ileum and rectum. The main function of this part of the intestine is to absorb water that has been delivered into the Malpighian tubule and therefore the cells in this part of the intestine are metabolically active.
Since plant feeding insects also have to handle compounds contained in the food that may be toxic to the insect an efficient cytochrome P-450 system has been developed, which will convert these compounds into water soluble products and excreted via the Malpighian tubule. The P-450 system is highly represented in the midgut.
The structure and function of the insect intestine makes it a strong candidate for studying intestinal drug uptake and could be a relevant model for highly efficient studies and early characterization, documentation and selection of compounds intended for oral administration.
It is an object of the present invention to use insect models that are aimed to reflect vertebrate absorption of drugs in the intestine or gut.